PURIFICATION OF IMMUNOGLOBULIN-E (IGE) ANTIBODIES FROM SERA WITH HIGHIGE TITERS

A three stage method for the ultrapurification of polyclonal IgE from human serum is reported using anion exchange chromatography followed b y monoclonal antibody based positive and negative affinity chromatogra phy. Following dialysis of 25-100 ml of serum (2.3-14 mu g IgE/ml, n = 4) against 0.05 M Tris pH 8, each specimen was subjected to diethylam inoethyl (DEAE)-cellulose chromatography (serum/matrix = 1/4). IgE was eluted with 0.05 M Tris, 0.05 M NaCl pH 8, yielding an IgE recovery o f 61-93%, with removal of similar to 90% of other serum proteins and a n IgE purity ([IgE]/[Igs]) of 0.1-1.1%. After adjusting to 0.1 M NaCl and concentrating similar to 30-fold, the eluted IgE was further purif ied by affinity chromatography using a panel of IUIS/WHO-documented mo use monoclonal anti-human immunoglobulin antibodies (alpha hIg-MAbs). First, the IgE-enriched DEAE-cellulose chromatography fraction was inc ubated in a batch mode with two alpha hIgE-Fc MAbs (HP6029, HP6061) co upled to CNBr-Sepharose, CL-4B. IgE was eluted with 0.05 M glycine pH 2.8 and immediately neutralized. The IgE recovery was 32-52% and IgE p urity was 72-97%. Silver-stained SDS-PAGE and noncompetitive solid-pha se two-site immunoenzymetric assays for total human IgA, IgE, IgG and IgM indicated that IgA, IgG and IgM were the only contaminants. Next, the IgE was concentrated 10-30-fold in the presence of 0.1% HSA. One I gE specimen was ultrapurified in a batch mode by negative selection ch romatography using three pairs of alpha hIg-MAbs (alpha hIgA: HP6111 HP6123; alpha hIgG: HP6017 + HP6046; alpha hIgM: HP6081 + HP6083) cou pled to CNBr-Sepharose, CL-4B. IgE purity increased from 91% to > 99.9 % with similar to 70% recovery of IgE for this step. The ultrapurified IgE antibody was shown to be functionally reactive for allergen and F c(epsilon)RI receptors on human basophils. We conclude that alpha hIg- MAbs are powerful tools to facilitate the affinity purification of fun ctionally active human IgE from serum; however, when the analyte is pr esent in low concentration, a carrier protein needs to be added to min imize non-specific loss of the material during this process.